JPH08269093A - Lectin complex,and method and probe for producing it - Google Patents

Abstract

A cytotoxic lectin complex having diminished non-specific toxicity comprising a cytotoxic lectin covalently bonded to a specific ligand comprising a disaccharide or higher polysaccharide derivative capable of binding specifically to an oligosaccharide-binding site on the lectin and a monoclonal antibody covalently bonded to the cytotoxic lectin complex.

Description

Detailed Description of the Invention

The present invention relates to a method for producing a cytotoxic lectin complex having reduced non-specific toxicity and a novel ligand useful in producing such a cytotoxic lectin complex.
A support complex or probe.

[0002]

BACKGROUND OF THE INVENTION There are several known cytotoxic lectins such as ricin, abrin, modesin, volkensin and biscumin, which kill eukaryotic cells very effectively. These lectins are heterodimeric proteins containing two subunits linked by a disulfide bond. One subunit, termed the A-chain, exerts cytotoxic activity by catalytic inactivation of the ribosome, while the other subunit, the B-chain, contains a specific binding site for galactose-terminated oligosaccharides. . Molecules containing oligosaccharide moieties are ubiquitous on the cell surface, so lectins are indistinguishable and therefore their cytotoxicity is non-specific. This property greatly limits the usefulness of selectively killing pathological or abnormal cells, such as tumor cells.

Conventionally, lectins are divided into two subunits, and only the A chain is bound to a monoclonal antibody to give a toxin having a specific activity, but such a complex product is significantly reduced as compared with that of all lectins. Shows toxicity.

Further, the oligosaccharide binding sites of lectins and concanavalin A were treated with a photoactivatable aryl azide derivative of mannose which specifically binds to the binding site of concanavalin A, and then exposed to ultraviolet rays to expose concanavalin A and polysaccharides. Blocking by forming a covalent bond with the derivative is described in J. Biochem. 78, 1013.
It is proposed by Beppu et al. As described in pp. 1019 (1975). The product retained activity at two of its four binding sites and exhibited reduced hemagglutination activity. Succinylated concanavalin A
Using Fraser et al., Proc, Nat, Acad. Sci. (US
A) 73, 790-794 (1976), and Thomas's Methods Enzymol. 46, 362-41.
On page 4 (1977), similar results are recorded by a similar operation. Similarly, Baenziger et al ., J. Biol.
Chem. 257, 4421-4425 (1982).
It has been described that the lectins concanavalin A, lysine and liver lectin are treated with a photoactivatable derivative of a suitable glycopeptide, and then light exposure results in the formation of a covalent bond between the lectin and the glycopeptide derivative. There is. However, in each case the glycopeptide derivative could only be included at 1-2%. It was clear that no attempt was made to determine the labeling efficacy of the lectin by measuring either cytotoxicity (for ricin) or hemagglutination (for concanavalin A). Houston by J. Biol. Ch
Similar procedures have been applied with photoactivatable derivatives of lysine and galactose as described in em. 258, 7208-7212 (1983). A
Although only the chain showed sufficient activity in cell lysates to inhibit protein synthesis, this product was found to be 280-fold less toxic to cells than untreated lysine.

Thorpe et al., Eur. J. Biochem. 140
Vol. 63-71 (1984), conjugating the monoclonal antibody to intact lysine and then fractionating the product by affinity chromatography isolates 20% yield fractions. In this case, it is stated that the monoclonal antibody accidentally blocks the oligosaccharide binding site of lysine, which reduces the ability of this complex to bind non-specifically to cells.

The variability in the final results obtained when photoactivatable oligosaccharides or monoclonal antibodies are used as blocking agents for the oligosaccharide binding sites of lectins is that the cytotoxic properties of the lectins are not reduced too much. Eliminated by the present invention, which effectively and reproducibly reduces non-specific toxicity.

The present invention is a method for producing a cytotoxic lectin with reduced non-specific toxicity, which comprises providing a ligand specific for an oligosaccharide-binding site on the lectin, the ligand being applied to a solid support. By covalently binding to form a probe and contacting the lectin with the probe by a specific interaction between the binding site and the ligand, and further forming a covalent bond between the lectin and the ligand. , Providing a complex of the ligand and the lectin, the ligand being covalently bound to the support. The invention also comprises the further step of breaking the covalent bond between the ligand and the support to release the complex containing the lectin covalently bound to the ligand. The invention also includes the additional step of forming a covalent bond between the complex and the monoclonal antibody. Other further features will be apparent from the description below.

The method of the present invention can be used with any lectin having cytotoxic activity such as ricin, abrin, modesin, volkensin or biscumin.

The solid carriers used in the present invention include crosslinked polyacrylamide and its derivatives such as aminoethyl polyacrylamide, derivatized porous glass beads, latex beads, polyvinyl alcohol beads and the like.
It may be any of the solid supports commonly used for affinity chromatography.

The ligand used in this method may be any compound that specifically binds to the oligosaccharide binding site of the lectin. Preferably, the ligand comprises a polysaccharide, such as a disaccharide or a higher polysaccharide, and preferably contains a galactose component for optimal binding capacity.

The covalent bond between the ligand providing the probe and the carrier can be formed by any conventional method. Any suitable heterobifunctional cross-linking agent, most known and commercially available, can be used. In some cases, the conjugate may remain bound to the carrier during subsequent use, but the crosslinker
It must be cleavable when subjected to selected conditions or reagents to allow it to separate from the carrier after the ligand-lectin complex is formed. Alternatively, one or both of the carrier and the ligand may be modified to include a functional group capable of reacting with the other to form a covalent bond between the ligand and the carrier,
Eliminates the need to use a separate crosslinker. The component that confers the ability to cleave the ligand from the carrier to which the ligand is covalently bound is preferably cleaved by reduction under selected conditions such that the lectin's own internal disulfide bond groups are not so rapidly cleaved. A disulfide group that can be cleaved, a thioether group that can be cleaved by aminolysis, preferably an azo group that can be cleaved by reduction with dithionite, about 36
It takes the form of an ortho-nitrobenzyl ester or ortho-nitrobenzyl carbamate that can be cleaved by light with a wavelength of 0 nm, and a vicinal glycol group that can be cleaved by oxidation with a periodate ester (or salt). You may

Similarly, the covalent bond between the ligand and the lectin is formed, for example, by conventional chemical methods by which proteins or polypeptides are linked to other compounds or groups by using a suitable cross-linking agent. be able to.

The covalent bond between the ligand and the carrier and the covalent bond between the ligand and the lectin can be respectively formed in two or more successive steps, if desired. For example,
2-Pyridyldithiopropionic acid can be reacted with a carrier such as aminoethyl-polyacrylamide to give a carrier having a 2-pyridyldithio group attached.
A ligand containing a disaccharide component denatures lactose to produce N
-(2'-Mercaptoethyl) lactoamine is formed and then the 2-pyridyl group on the carrier is replaced by disulfide exchange and the amine residue of lactoamine is then used as a functional group to form a covalent bond with the lectin. can do. This is because the reactivity of one chlorine atom is pH such as 2,4-dichloro-6-methoxytriazine.
It can be achieved by using a bifunctional crosslinker which is large in 8, the reaction being complete in minutes. The remaining chlorine atoms are more gradual, 2 at pH 8.5 and above.
Reacts with amino groups in 4 hours. This crosslinker is first reacted with the amino residues of lactoamine at pH 8 and then correctly positioned by specifically binding the lectin to the galactose component of lactoamine, then at higher pH it is reacted with the amino groups of lectins. .

The second cross-linking agent does not need to be cleavable because it is desirable that the ligand is permanently bound to the lectin and blocks the latter oligosaccharide binding site. In another embodiment, the ligand itself can be chemically modified to have a lectin-reactive functional group to form a covalent bond between them.

When the ligand itself is modified to have two functional groups, one functional group can form a covalent bond with the carrier and the other can form a covalent bond with the lectin. The two functional groups are preferably different in reactivity or reaction conditions so that the ligand binds to the carrier under a certain set of conditions and binds to the lectin under another set of conditions. In this case, the ligand itself
It is considered to be a heterobifunctional cross-linker containing a disaccharide or a polysaccharide that can specifically bind to the oligosaccharide binding site of a lectin.
The two functional groups included in the ligand may be similar to those present in conventional heterobifunctional crosslinkers.

The ligand is bound to a solid support to form a probe, the lectin is placed on the probe by specific binding and then the ligand-lectin complex is formed, and then, if desired, the ligand-support binding is carried out by conventional procedures. May be separated from the carrier by cleavage.

The complex is either free or remains bound to a solid support and has oligosaccharide binding sites blocked by a ligand, resulting in non-specific or cell-specific binding to cells. It no longer exhibits non-selective binding and loses most of its non-specific toxicity. The complex is then covalently linked to a selected monoclonal antibody that exhibits specific immunological binding capacity for specific cells,
It can be treated to show specificity for the particular cell of interest to be attacked. Covalent attachment of the complex to the monoclonal antibody can be carried out before or after being cleaved or cleaved from the carrier using conventional manipulations or cross-linking agents such as those described in Thorpe et al . , Loc. Cit .

In a preferred embodiment, as shown in Example 1, the complex can be bound to the carrier by a ligand via a disulfide group which can be cleaved by reduction. The thiol residue left on the ligand is then used as a functional group to form a covalent bond with the monoclonal antibody. For this purpose, any suitable bifunctional crosslinker capable of binding the thiol group to the amino group or other groups of the monoclonal antibody may be used. Preferably, a functional group that reacts with a thiol group to form a covalent bond is introduced into the monoclonal antibody so that it can react directly with the functional group of the complex. For example, if the functional group on the complex is a thiol, the monoclonal antibody is reacted with a reagent such as succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to allow the maleimide group to be present on the antibody. May be.

In another preferred embodiment, as shown in Example 2, the ligand (and the resulting lectin-
Ligand complex) is an ortho-capable of being cleaved by radiation.
The nitrobenzyl carbamate is attached to the carrier via a photocleavable group and a bifunctional cross-linking reagent is used to form a radiation stable covalent bond between the lectin and the ligand of the ligand-carrier probe. It

In a further preferred embodiment,
As shown in Example 3, the ligand is bound to the carrier by sodium dithionite via an azo group that can be cleaved by preferential reduction, while the lectin-ligand covalent bond provides an inactive bond to such reduction. It is formed by the bifunctional crosslinker that forms.

The following specific examples are intended to illustrate the method of the present invention in more detail and not to limit the scope of the invention.

[0022]

【Example】Example 1 Preparation of modified carrier InmanMethods Enzymology 34, 30-58 (1
974) and amino in the form of solid beads.
Ethyl polyacrylamide P-150 (B_Ten-Rad)
Is a water-soluble carbodiimide in an aqueous solution of ammonium chloride.
Treated by reacting with any residual carbo
The xyl group was capped. Then the beads, then pyridyldithio
By functionalizing with a group
As shown by the reactions described above, disulfide linkages
The position of attachment of the ligand via the tether was given. For this purpose
For the purpose of carboxy-capped-aminoethylpolyol
CrylamideThree(Filled beads 30 ml) suspension and 0.
To 10 ml of 1M sodium chloride solution, add dimethylforma
2-Pyridyldithiopro in 10 ml of amide and 1 ml of water
Pionic acid (P_y -S-S-C₂ H_Four -CO₂ H) 0.0
1 g (5 mmol) was added. Add diluted hydrochloric acid and mix.
The pH of the compound was adjusted to 4.7 and 1-ethyl-3- (3-
Dimethylaminopropyl) carbodiimide hydrochloride
Containing 1.15 g (6 mmol) of EDC (HCl)
Mixed with a suspension of 5 ml of water. Stir at room temperature for 7 hours
And then carbodiimide 0.766 g (4 mmol)
Was added and the pH of the mixture was readjusted to 4.7 with hydrochloric acid. Room
After shaking at warm temperature for 4 days, remove the liquid from the suspension and
beadsFourWith 0.2M sodium chloride solution for a long time
I washed it. Excess 2-pyridyldithiopropionic acid
In order to ensure complete removal of the
It was acidified with and extracted with 5 ml of ethyl acetate. beadsFour
With 100 mM sodium phosphate buffer, pH 7.0
Washed. When treated with excess dithioerythritol
The cleaning is carried out with 2-pi
Lysine thione is shown at 0.13 a at 343 nm.
u. Continue until it shows an absorbance of less than.

The surface concentration of the dithiopyridyl group in the column of the modified beads 4 was determined by measuring the sample of the beads in excess of 10.
It was measured by reacting with dithioerythritol (DTE) in 0 mM sodium bicarbonate solution for 30 minutes at room temperature. The beads were then repeatedly washed with 0.2 mM sodium chloride solution and the amount of 2-pyridinethione in the wash solution [T. Stu] determined by absorbance at 343 nm.
Chbury et al . , Biochem. J. , 151, 417-432 (1975)] was found to correspond to a concentration of 38 micromolar per ml of packed beads.

Polymer beads containing 2-pyridyldithio 4
To cap any excess amino groups present above, the beads (27 ml) were suspended in 50 ml of 100 mM sodium bicarbonate solution, to which was added 4.8 g (5.2 mmol) methyl chloroformate at room temperature. It was After 5 minutes, excess chloroformate was removed by extraction with 20 ml of ethyl acetate and the modified gel beads at pH 7.0.
Washed with 100 mM sodium phosphate buffer.

Preparation of Ligand The ligand was prepared as shown in the upper left part of FIG. Water 3
9.72 g (1 of α-lactose monohydrate 1 in 6 ml)
3.5 mmol) was dissolved. To the above solution, 3.68 g (13.5 mmol) of cystamine diacetate and 1.70 of sodium cyanoborohydride in 72 ml of methanol.
g (27.0 mmol) solution. P of the mixture
After adjusting H to 6.2 with acetic acid, the solution was stirred at room temperature for 36 hours, during which methanol was removed by evaporation, the pH of the residual aqueous solution was adjusted to 5.0 with acetic acid and the solution was adjusted to pH.
It was passed through a 360 ml column of carboxymethylcellulose equilibrated in 5.0 mM 2 mM pyridinium acetate buffer. After washing the column with 2 volumes of equilibration buffer,
The bound material was eluted with 0.1 M aqueous ammonia solution. The solution containing the eluate was evaporated to dryness, and the solid content was 200 ml of water.
Redissolved in dithioerythritol (DTE) at room temperature
2.0 g (13.5 mmol) was added. After standing for 1 hour, the resulting solution containing a mixture of N- (2'-mercaptoethyl) lactoamine 2 and cysteamine was added to 1
It was passed through a 370 ml column of carboxymethylcellulose in 10 mM triethylammonium bicarbonate buffer, pH 7, containing mM DTE. Use the same buffer 5
Wash with 00 ml, then p containing 1 mM DTE
It was developed with a linear gradient (1 liter + 1 liter) solution of H7 in 10-100 mM triethylammonium bicarbonate. Repeated evaporation from water and freeze-drying gave 3.85 g (35% of theory) of N- (2'-mercaptoethyl) lactoamine ligand 2 as a white powder.

Immediately before using the ligand 2 prepared and purified as described above, 1.5 g (3.7 mmol) of the ligand was dissolved in 10 ml of water at pH 8 to give DTE154.
All dimers formed between two molecules of lactoamine were subjected to reduction treatment by adding mg (1 mmol) and allowing the mixture to stand at room temperature for 1 hour. The pH was then reduced to 5.2 with acetic acid and the solution was passed through a 100 ml column of carboxymethylcellulose in 2 mM pyridinium acetate buffer, pH 5.2. The column was washed with 200 ml of the same buffer until all DTE was removed. Pure N-
(2'-mercaptoethyl) lactoamine 2 was added to 0.1
It was eluted with M hydrochloric acid in the form of the free thiol. Analysis by Ellman's reagent showed that the solution contained 3.2 mmol of thiol (85% of theory).

Preparation of the Ligand-Carrier Probe 50 ml (3.2 mmol) of a pH 5 aqueous solution of lactoamine was added to 27 ml of gel beads at room temperature to give a suspension of pH 6.5, which was shaken at room temperature for 15 minutes and then 0. Repeated by sequential centrifugation and decantation steps using a 2M sodium chloride solution, then the washings were substantially 343n of 2-pyridinethione byproduct.
ligand 2 by washing until it shows that it has been removed as indicated by measurement of the absorbance at m.
Was covalently bound to the previously prepared capped and activated polyacrylamide gel beads 4. The generated beads are
It had a surface lactose concentration of 38 mM, in the form of probe 5, which contained a ligand covalently attached to the polymer beads.

In order to covalently attach lysine to the ligand-carrier probe 5, a suspension containing 10 ml of probe in 10 ml of a solution of 100 mM sodium bicarbonate was added to the suspension.
360 mg of the triazine reagent in 0 ml dioxane (2
The probe was activated with the difunctional crosslinker 2,4-dichloro-6-methoxytriazine by adding a solution containing (mMole). After the suspension was swirled for 1 minute, it was extracted with 4 ml of diethyl ether to remove excess triazine, the activated probe beads 6 and then the pH.
100 mM sodium phosphate buffer 7.0 was equilibrated and loaded onto the column.

Preparation of Lectin-Ligand-Carrier Complex Activated lysine in the form of 2.5 ml of a solution containing 2.1 mg / ml lysine in 0.01 M sodium phosphate buffer pH 7 containing 0.15 M NaCl. Applied to a modified probe column. The column was then washed with 3 column volumes of 50 mM triethanolamine hydrochloride buffer pH 8.6 to form a covalent bond between the lysine and the second functional chlorine group of the triazine crosslinker. PH at room temperature
After standing at 8.6 for 24 hours, covalently bound lysine-
The column was washed with 3 volumes of 0.5 M galactose solution at pH 7 to remove any lysine that did not form a ligand complex.

Cleavage of lectin-ligand complex from carrier
Covalently bonded ricin was prepared as previously sectional above - ligand complex was cleaved from the support by cleaving the disulfide group between the ligand and the polymer. It was found that this disulfide group was more easily cleaved or cleaved by reducing with 10 mM DTE at pH 7 than the disulfide group connecting the A-chain and B-chain of lysine itself. DTE with a pH value of 7 to 8 was investigated at various concentrations.
Optimum result of minimum lysine chain cleavage is DTE concentration 10m
M, pH 7.0, obtained for 45 minutes at room temperature, theoretical 3
Yield 6%. The free complex thus obtained did not bind to asialofetuin, demonstrating that the oligosaccharide binding site of lysine was blocked.

The lysine-ligand complex prepared as described above was affinity-purified on a column of Concanavalin A-Sepharose in 100 mM sodium phosphate buffer, pH 6. The conjugate binds to concanavalin A via the carbohydrate chain of the lysine component, thereby eliminating contaminants, especially low molecular weight thiols, which hinder the conjugation reaction. Lysine-ligand complex was added at pH 6 containing 1M methyl α-D-mannopyranoside at pH 6.
12m solution eluting with 0mM sodium phosphate buffer containing 150-200μg lysine-ligand complex
yielded l. Product trials have shown that the A chain suppresses protein synthesis in reticulocyte lysates to the same extent as the natural lysine-derived A chain.
The desired modification of this material was confirmed by passage through a column of immobilized asialofetuin and was shown to have an association constant for lysine of approximately 0.1 μM. By this criterion, lysine binding sites were considered blocked.

Monocloner of lectin-ligand complex
Murine monoclonal anti-CALLA commercially available from Coulter Incorporated, Hialia, Fla., In order to confer on the covalently bound lysine-ligand complex to the antibody the desired specificity for the selected cells. Antibody J5
Was bound. For compounding, Lambert
Et al., J. Biol. Chem. 260, 12035-12041.
J5 was purified as described in (1985) and then modified with SMCC.

A solution of the lysine-ligand complex as described above was then purified and modified J in pH 7 buffer.
It was mixed with 5 antibodies and allowed to stand for 1 hour at 4 ° C. 1
After time, it was found that the reaction mixture contained a covalently bound conjugate between the monoclonal antibody and the lysine-ligand complex, as well as an excess of monoclonal antibody and some unreacted lysine-ligand complex.
Indicated by E. The reaction mixture is then brought to pH 7.0.
Affinity purification was performed with Protein A-Sepharose in the sodium phosphate buffer solution described in 1. above to remove unreacted lysine-ligand complex. This step also removed the methyl α-D-mannopyranoside introduced earlier, thereby allowing the column of concanavalin A-Sepharose to be used as a further purification step. Protein A
-The material eluted from the Sepharose column contains the conjugate and excess monoclonal antibody, which is then applied to Concanavalin A-Sepharose, where it binds the conjugate through the carbohydrate chain of the lysine-ligand complex as before. Let Excess monoclonal antibody was then removed by extensive washing and bound conjugate at pH 7.0.
Eluted with 1M methyl α-D mannopyranoside in 100 mM sodium phosphate buffer.

Namalwa cells, on average 60,000 CALLA per cell and 600 for lysine,
Burkit (Burkit
The t's) lymphoma line was used to test the cytotoxicity of both the ricin-ligand complex and the complex bound to the monoclonal antibody. The ricin-ligand complex was found to be about 10 times less toxic than native ricin. This reduction in non-specific toxicity was shown to be fully active when the A chain was released from the complex (see above), B
By blocking binding sites on the chains. When the lysine-ligand complex is bound to J5, its toxicity is CALL.
It was increased 3-fold for target cells representing A and its toxicity was reduced 5-fold for non-target cells lacking CALLA. Most significantly, the toxicity of the conjugate was reduced 3-fold by adding a saturating amount of J5, clearly indicating that the antibody confers some specificity on the modified toxin.

Example 2 The reaction scheme for making the probe of this example is shown in FIG. Modified Carrier Preparation A suspension (13 ml) of carboxycapped aminoethyl polyacrylamide P-1503 (26 ml of packed beads) prepared as described in Example 1 in 0.1 M sodium bicarbonate was added to dioxane. 1 in (6 ml)
-(5-Maleimidomethyl-2-nitrophenyl) -ethyl chloroformate 7 (1.02 g, 3.48 mmol) was added. After vigorous shaking for 5 minutes, methyl chloroformate (5 ml) was added to cap any excess amino groups and shaking was continued for another 5 minutes. Then, the modified polyacrylamide gel beads 8 were collected by filtration, and the modified polyacrylamide gel beads 8 were collected using a mixture of 0.1 M sodium phosphate buffer of pH 7.0, 0.1 M sodium phosphate buffer of pH 7.0 and dimethylformamide (1: 1). ,
Volume / volume) and dimethylformamide, and then the same solution in reverse order.

Preparation of ligand-carrier probe Modified polyacrylamide beads 8 (packed beads 24
ml) was suspended in 0.1 M sodium phosphate buffer pH 7.0 (10 ml) and the ligand N- (2′-mercaptoethyl) lactoamine 2 (700 mg, 1 prepared as described in Example 1). PH of 0.74 mmol)
It was treated with an aqueous solution of 7.0. The mixture was shaken overnight,
Probe 9 containing the ligand covalently bound to the polymer beads by filtration on a Buchner funnel.
Was recovered and washed thoroughly with a sodium phosphate buffer of pH 7.0. A small column was prepared with a sample of ligand-carrier probe 9 (0.3 ml) and the specific binding capacity of the probe beads for lysine was measured. It was found that at pH 7.0 1 ml of probe beads bound 1.0 mg excess of lysine in a specific manner. All specifically bound lysine could be eluted with a buffer containing 0.2M lactose.

The column of probe beads 9 was activated with the bifunctional cross-linking agent 2,4-dichloro-6-methoxy-triazine by the following method. To a suspension of polyacrylamide beads containing lactose ligand (10 ml) in 0.1 M sodium bicarbonate (10 ml) was added 2,4-dichloro-6-methoxytriazine (0.24 g, 1) in dioxane (6.6 ml). Solution of 0.34 mmol) was added. The suspension was shaken vigorously for 1 minute and then extracted with diethyl ether (3 x 5 ml) to remove excess triazine. Activated probe beads 10
Was then equilibrated with 0.1 M sodium phosphate buffer pH 6.5 and loaded into the column.

Preparation of lectin-ligand-carrier complex A solution of lysine (p containing 0.15M sodium chloride
10 mg) in 5 ml of 0.01M sodium phosphate buffer of H7.0) was passed twice through the activated probe column, which was then passed through three column volumes of 0.0 pH 8.6.
Washed with 5M triethanolamine hydrochloride and left at ambient temperature for 24 hours. Bound but not covalently cross-linked lysine was removed from the beads by washing with 0.1 M sodium phosphate buffer, pH 7.0 containing 0.2 M lactose.

Separation of lectin-ligand complex from carrier
The lysine retained by covalent bond with the dissociated ligand was separated from the carrier beads by photolysis in the following manner. The beads were washed with 0.1 M sodium phosphate buffer (10
ml) was used to transfer from the column to a glass petri dish, where the suspension was allowed to form a layer with a thickness not exceeding 0.5 cm. This suspension is then treated with a black line longwave UV lamp (B-100 to break the bond between the ligand and carrier).
Type A, Ultraviolet Products, Inc., San Gabriel, Calif., Emission peak 365 nm, 1
Intensity at 5 cm of about 1.1 mW / cm ² ) to 15 c
Irradiation was carried out for 10 minutes at a distance of m. The irradiated suspension was poured back into the column and the beads were thoroughly washed with 0.1 M sodium phosphate buffer, pH 7.0. Combined wash solution containing lysine-ligand complex separated from carrier (5
0 ml) was passed through a column of asialofetuin-TSK (2 ml, binding capacity for lysine 4 mg / ml) to remove possible traces of lysine impurities. The final solution was concentrated by ultrafiltration (YM-10 semipermeable membrane, Amicon, Inc., Danvers, MA) to 2 ml,
Then sodium chloride (150 mM) and EDTA (1 m
Biogel P- equilibrated in 0.05 M triethanolamine hydrochloride buffer, pH 8.0 containing M) and
Passed through 6 small columns, 1.2 mg of pure lysine-ligand complex in 2.8 ml of buffer was obtained.

Lectin-ligans for monoclonal antibodies
In order to covalently attach the covalent complex of the antibody conjugate to antibody J5, the heterobifunctional crosslinker 2-iminothiolane hydrochloride (Peace Chemical Co., Illinois, Ill.) Was prepared as described in detail below.
Lockhod) was used to introduce a sulfhydryl group into the complex to give a heterobifunctional cross-linking agent
A maleimide group was introduced into a monoclonal antibody using (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (Peace Chemical Co., Rockford, IL).

Solution of lysine-ligand complex (2.8 m
l) was cooled on ice and treated with 0.5M 2-iminothiolane hydrochloride solution (pH 44) (0.044 ml) to give 2-
The final concentration of iminothiolane was 8 mM. P containing sodium chloride (50 mM) and EDTA (1 mM)
The reaction was stopped after 90 minutes on ice by gel filtration through a column of Biogel P-6 equilibrated with 5 mM Bis-tris-acetate buffer in H5.8. Thus
An average of 0.8 thiol groups was introduced per molecule of lysine-ligand complex.

A solution of J5 in 0.1 M sodium phosphate buffer, pH 7.0 (3 mg J5 in 1.5 ml).
And 12 equivalents of S in dry dioxane (0.02 ml)
MCC (0.075 mg) was added. After incubation for 30 minutes at 30 ° C., the reaction solution is placed at 4 ° C.
A modified J5 having an average of 1.5 maleimide groups per antibody molecule was obtained by passing through a column of Sephadex G-25 in.

Modified lysine-ligand complex solution and modified J
The 5 solution was mixed and the pH was adjusted to 7.0 by adding 0.5 M triethanolamine hydrochloride (0.028 ml) at pH 8.0. After standing overnight at 4 ° C., iodoacetamide was added to a final concentration of 5 mM, and the solution was concentrated to about 2 ml by ultrafiltration (YM-10 filter, Amicon). Purification was performed by gel filtration through a column of Biogel P-300 (1.5 x 96 cm) in phosphate buffered saline. Both the lysine-ligand complex and the complex bound to the monoclonal antibody showed reduced non-specific toxicity properties similar to the product of Example 1.

Example 3 Ligands are bound by azo functional groups and can be separated by treatment with 0.5M sodium dithionite at pH 8.5 (eg Cohen).
"Methods Enzymol.", Vol. 34 (1974).
Years), pp. 102-108)). A reaction formula for producing the probe is shown in FIGS. 3A and 3B.

Ligand Preparation To a stirred solution of -lactose (4.0 g, 11.1 mmol as monohydrate) in water (40 ml) was added aniline (2.07 g, 22. 2 mmol) and a solution of sodium cyanoborohydride (0.7 g, 11.1 mmol) in methanol (6 ml) was added. The pH of the mixture was adjusted to 6.5 with acetic acid and the solution was stirred at room temperature.

After 15 hours the methanol was removed by evaporation, acetone (12 ml) was added to destroy excess sodium cyanoborohydride and the solution was stirred at room temperature for 1 hour. The solution was then evaporated to dryness,
The residue was redissolved in water (75 ml), the solution was adjusted to pH 5.5 with acetic acid and applied to a column of Amberlite IRA-118H ion exchange resin (60 ml) equilibrated in water. Water this column (600m
Washing with l) and elution of the sugar with a 1 M solution of ammonium bicarbonate gave 5.4 g of a white solid containing N-phenyllactoamine ligand 13 contaminated with some salt.

Preparation of modified carrier Carboxy-capped aminoethyl polyacrylamide P-1503 (12 ml of packed beads) 0.2M
To a suspension in NaCl (total volume 20 ml), THF
P-Nitrobenzoyl azide (576 m in (5 ml)
g, 3 mmol) and triethylamine (304 mg, 3
Mmol) and was added. The gel was shaken gently for 20 minutes, then another 304 mg of triethylamine was added.
(3 mmol) was added. After shaking the suspension for an additional 25 minutes, the p-nitrobenzoyl substituted carrier beads were mixed with a mixture of THF and 0.2 M NaCl solution (1:
1, volume / volume), formamide and 0.2 M NaC
It was washed successively with 1 solution.

Next, the beads were treated with 100 mM NaHC.
Suspended in a solution of O ₃ (10 ml) and shaken vigorously with methyl chloroformate (2 ml) to cap any excess amino groups. The suspension was extracted with diethyl ether (2 x 15 ml) to remove excess chloroformate and the beads were adjusted to pH 7.0 with 1
It was washed with 00 mM sodium phosphate buffer. pH 7.
To a suspension of the gel in 0 of 100 mM sodium phosphate buffer (final volume 20 ml) was added sodium dithionite (2.61 g, 15 mmol). The suspension was then held at 55 ° C in a water bath, periodically removed and shaken. After 45 minutes, the p-aminobenzoyl substituted carrier gel beads were washed with a 0.2 M NaCl solution. A suspension of these modified polyacrylamide gel beads (5 ml of packed beads) in 1M-HCl (final volume 10 m
l) at 0 ° C. with sodium nitrite (69 mg, 1
(Mmol) cold aqueous solution (1 ml) was added. The suspension was gently shaken at 4 ° C. for 20 minutes, then the beads were washed rapidly with cold 0.2M NaCl solution to obtain p-diazobenzoyl substituted carrier beads 12.

Preparation of Ligand-Carrier Probe A solution of N-phenyllactoamine ligand 13 (810 mg, 1.9 mmol) in saturated sodium borate solution (10 ml) was added to the cooled diazo modified carrier 12 which was immediately added. It turned deep red. The suspension was gently shaken overnight at 4 ° C., and then washed thoroughly with 0.2 M NaCl solution to separate the deep red ligand-carrier probe 14 in which the ligand was covalently bound to the carrier via the azo group. Let Part of the ligand-carrier probe 14 (0.50 m
l) was used to produce a small column with which a specific binding capacity of 0.5 m / ml packed beads was obtained.
It was shown to be g. It was demonstrated that lysine was specifically bound by showing that bound lysine can be eluted in two ways. The specific interaction of lysine-ligand can be disrupted by competition with high concentration of galactose, and the ligand-carrier bond is cleaved by treatment with 0.2-0.5M sodium dithionite solution at pH 8. be able to. In both cases, lysine or lysine-ligand conjugate was eluted quantitatively from the column. As a further proof that treatment with dithionite releases lysine by cleavage of the ligand from the carrier, this gel immediately loses the deep red color due to the development of the azo group.

The ligand-carrier probe 14 was activated by using the bifunctional cross-linking agent 2,4-dichloro-6-methoxytriazine by the following method. To a suspension (20 ml) of ligand-carrier probe beads 14 (volume of packed beads 5 ml in 100 mM sodium phosphate buffer, pH 7.0) in dry DMF, 2 in dry DMF (10 ml).
4-dichloro-6-methoxytriazine (1.8 g, 1
0.0 mmol) solution was added. The suspension was gently stirred at room temperature for 22 hours. The activated probe beads 15 were then added to DMF, DMF-100 mM sodium phosphate buffer pH 7.0 and finally pH 7.
0 was washed successively with 100 mM sodium phosphate buffer and loaded into the column.

Preparation of Lectin-Ligand-Carrier Complex A solution of lysine (p containing 0.15 M sodium chloride
5.7 mg) in 3 ml of 0.01 M sodium phosphate buffer of H7.0) was applied to the activation probe column 15,
This flow-through was applied twice. The column was washed with 3 column volumes of 0.05 M triethanolamine hydrochloride, pH 8.6, and left at ambient temperature for 24 hours. Bound but not covalently cross-linked lysine was removed by washing the column with 0.1 M sodium phosphate buffer, pH 7.0 containing 0.5 M galactose. The lysine-ligand complex was then separated from the carrier by cleaving the covalent bond in the following manner. The column was washed with 0.05 M triethanolamine hydrochloride buffer, pH 8.6 to remove residual galactose, and then with 0.2 M sodium dithionite solution, pH 8.0 containing 0.3 M sodium chloride. . The beads are then transferred to a separation container and the pH
Stir for 20 minutes with a solution of 8.0 0.2 M sodium dithionite and 0.3 M sodium chloride. The suspension was filtered and the beads treated with dithionite two more times in a similar manner. The dithionite solution was stored and 12 ml of solution was obtained containing about 0.8 mg of the lysine-ligand complex released from the carrier. This complex can be covalently attached to a monoclonal antibody such as J5 using a conventional bifunctional crosslinker.

Lysins can be replaced by other lectins with similar results. The lectin-ligand-monoclonal antibody complex can be used to kill selected cells in vivo as well as in vitro.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a reaction generated by an activated ligand-carrying probe of Example 1.

FIG. 2 is a schematic diagram showing a reaction generated by the activated ligand-carrying probe of Example 2.

FIG. 3 is a schematic diagram showing a reaction generated by the activated ligand-carrying probe of Example 3.

Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location G01N 33/577 A61K 37/46 ADU (72) Inventor Brettler, Walter A. USA 02146 Brooklyn, Massachusetts , Mason Terrace 197 (72) Inventor Lambart, John Em. United States 02146 Massachusetts, Brookline, Linden Place 21 (72) Inventor Dalarkao, Linda Jay. United States 02130 Massachusetts, Jamaica Plain, One Regent Circle ( (No address)

Claims (23)

[Claims] 1. A blocked lectin, wherein said oligo of said lectin is such that it blocks one or more of the oligosaccharide binding sites of said lectin by a non-photoactivatable reactive group present on each ligand. A blocked lectin as described above comprising one or more affinity ligands that are specifically covalently bound to the sugar binding site. 2. The blocked lectin of claim 1, wherein at least one of said covalently bound affinity ligands comprises a moiety capable of covalently binding said ligand to a monoclonal antibody. 3. The blocked lectin according to claim 1, wherein the lectin comprises a component capable of covalently binding the lectin to a monoclonal antibody. 4. The blocked lectin according to claim 1, 2 or 3, wherein the affinity ligand comprises a sugar derivative specific for a cytotoxic lectin. 5. The blocked lectin according to claim 1, 2 or 3, wherein the affinity ligand comprises a sugar derivative specific for lysine. 6. A blocked lectin according to any one of claims 1, 2 or 3 wherein the affinity ligand comprises at least one terminal galactose derivative. 7. The blocked lectin according to any one of claims 1, 2 or 3, wherein the reactive group that covalently bonds the ligand to the lectin is capable of cross-linking a protein. 8. A blocked lectin according to any one of claims 1, 2 or 3 wherein said one or more affinity ligands comprises N- (2'-mercaptoethyl) lactoamine. 9. The blocked lectin according to claim 1, 2 or 3 wherein said one or more affinity ligands comprises N-phenyllactoamine. 10. The blocked lectin according to claim 1, 2 or 3, wherein the lectin is selected from the group consisting of ricin, abrin, modesin, volkensin, and derivatives thereof. 11. The blocked lectin according to claim 10, wherein the cytotoxic lectin is ricin. 12. (1) (a) 1 through a non-photoactivatable reactive group present on an affinity ligand
By covalently attaching one or more affinity ligands to a solid support, and (b) to form a reactive group capable of covalently attaching at least one said one or more affinity ligands to a lectin. To produce an affinity carrier (however, the order of the covalent bond of the (a) and the activation of the (b) does not matter), (2) the oligosaccharide bond of the lectin to the lectin. Binding at least a portion of one or more activated affinity ligands having an affinity for the site, and (3) by covalently linking the ligand to the lectin through a reactive group on the ligand. Present on each ligand containing a step that blocks one or more oligosaccharide binding sites of the cytotoxic lectin Blocked by the reactive group including the one or more affinity ligands specifically covalently linked to the oligosaccharide binding site of the lectin so as to block one or more of the oligosaccharide binding site of the lectin. Method for preparing lectin. 13. The method according to claim 12, wherein the covalent attachment of the (a) is performed prior to the activation of the (b). 14. The method of claim 12, comprising the step of cleaving the covalent bond between the one or more ligands and the solid support to release the blocked lectin from the solid support. 15. A method comprising recovering the blocked lectin from an unblocked lectin.
The method according to 4. 16. The affinity ligand comprises a sugar derivative specific for a cytotoxic lectin.
The method according to any one of 3, 14, or 15. 17. The method of any one of claims 12, 13, 14 or 15 wherein the affinity ligand comprises a sugar derivative specific for lysine. 18. The method of claim 12, wherein the affinity ligand comprises at least one terminal galactose derivative.
The method according to any one of 3, 14, or 15. 19. The method of any one of claims 12, 13, 14 or 15 wherein the reactive group that covalently attaches the ligand to the lectin is capable of cross-linking a protein. 20. The method of any one of claims 12, 13, 14 or 15, wherein the one or more affinity ligands comprises N- (2'-mercaptoethyl) lactoamine. 21. The method of claim 12, wherein the one or more affinity ligands comprises N-phenyllactoamine.
The method according to any one of 14 or 15. 22. The method of any one of claims 12, 13, 14 or 15 wherein the lectin is selected from the group consisting of lysine, abrin, modesin, volkensin, and derivatives thereof. 23. The lectin is ricin.
The method described in.